DESCRIPTION (adapted from applicant's description): The Na/K pumps establish the cell's transmembrane gradients in Na and K. These are essential for excitability, and the energy stored in the Na-gradient is used by Na/Ca exchange to maintain Ca homeostasis. Moreover, the pump generates an outward current that directly affects the cardiac action potential. Changes in Na/K pump activity can lead directly to arrhythmias, or through effects on Na/Ca exchange, lead to Ca overload and sudden death. Previous studies by the applicant have shown that guinea pig ventricular myocytes co-express the alpha1-and alpha2-isoforms, which have isoform specific, Ca-dependent coupling to autonomic input. The general questions addressed in this proposal are: What is the physiological purpose of this elaborate regulatory scheme, and how are the different signal transduction cascades coupled to each isoform? Preliminary data suggest the following hypotheses: Beta-adrenergic effects on the amount of Na/K pump current are through cycling of vesicles containing the alpha1-isoform; effects on voltage dependence are through direct phosphorylation and Ca-binding. The alpha1-isoform is regulated primarily for Ca homeostasis: the alpha2-isoform is regulated primarily to shape electrical activity. Aims 1, 2 & 3 are to investigate the coupling of signal transduction cascades to specific isoforms. Autonomic induced changes in membrane capacitance will be correlated with changes in pump current, while interfering with vesicle trafficking. The phospho-state of the isoforms will be determined using P-32 labeling and phospho-specific antibodies. Aims 4, 5 & 6 are to investigate the role of autonomic regulation of pump current in Ca homeostasis. The whole cell patch clamp technique will be used to characterize the Na/Ca exchanger and the Ca-dependence of the Na/K pump. Computer simulations will help to understand the interactions of these two transport systems with intracellular Ca. Aims 7 & 8 are to investigate how autonomic regulation of pump current directly affects electrical activity. The whole cell patch clamp will be used to inject current into ventricular action potentials, thus, mimicking the direct effects of autonomic mediated changes in pump current. It will also be used to characterize regulation of pump current in SA node pacemaker cells and determine if regulation of pump current (particularly the alpha2 isoform) affects pacemaking.